Research Article
Leukotriene D4 Requires PKC α- Akt Signaling Pathway to Inhibit Na+- Dependent Alanine Cotransporter (ASCT1) in Enterocytes
Jamilur R. Talukder1*, Jaleesa Wright1, Antara Jaima2 and Deja McIntosh1
1Department of Biology, LeMoyne-Owen College, 807 Walker Avenue, Memphis, TN 38126, USA
2Boston University, One Silber Way, Boston, MA 02215, USA
- *Corresponding Author:
- Jamilur R. Talukder
Department of Biology
LeMoyne-Owen College
807 Walker Avenue, Memphis
TN 38126, USA
Tel: +1-(901) 435-1396
Fax: +1-(901) 435-1424
E-mail: Jamil_Talukder@loc.edu
Received date: December 06, 2015Accepted date: December 19, 2015, Published date: December 26, 2015
Citation: Talukder JR, Wright J, Jaima A, McIntosh D (2015) Leukotriene D4 Requires PKCα- Akt Signaling Pathway to Inhibit Na+-Dependent Alanine Cotransporter (ASCT1) in Enterocytes. J Gastrointest Dig Syst 5:364. doi:10.4172/2161-069X.1000364
Copyright: © 2015 Talukder JR, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Varieties of inflammatory cytokines are produced during chronic enteritis including inflammatory bowel disease (IBD), that leads to malabsorption of nutrients and diarrhea. The inflammatory mediators are produced from different tissues of the body. Arachidonic acid metabolite derived via Lipoxygenase pathway, leukotriene D4 (LT) inhibits Na+-dependent alanine (Ala) cotransport (ASCT1, solute carrier, slc1a4) in the apical membrane of enterocytes by decreasing the affinity of cotransporter. However, the intracellular mechanism of LT-mediated inhibition of ASCT1 activity is unknown.
Objective:To investigate the intracellular mechanism of leukotriene D4 (LT) mediated inhibition of ASCT1 activity in enterocytes.
Methods: Rat intestinal epithelial cells (IEC-6) were grown on transwell plates. [3H]-Ala uptake was measured in 10 days postconfluent cells using a scintillation counter. IEC-6 cells were treated with different inhibitors in 8 days postconfluent to intercept different checkpoints of pathways for LT-mediated inhibition of ASCT1 activity. Intracellular Ca2+ and cAMP levels were measured. Immunoblotting, qRT-PCR, and immunocytochemistry were performed following the standard protocols.
Results: LT treatment increased more than 2.5-fold [(cAMP)i] and [(Ca2+ )i]. PKA, PKC-δ and -θ inhibitor did not reverse the LT-mediated inhibition of ASCT1 activity. However, PKC-α inhibitor antagonized LT effect on ASCT1 activity. Further downstream of PKC-α pathway, tyrosine kinase (Akt) inhibitor also reversed LT-mediated inhibition of ASCT1 activity. Immunoblotting, qRT-PCR, immunocytochemistry, and Kinetics studies demonstrated that the mechanism of decreased affinity of ASCT1 by LT was due to decrease in affinity of ASCT1 for Ala transport that was restored by Akt inhibitor.
Conclusion: Therefore, we conclude that LT inhibits ASCT1 activity by decreasing the affinity of ASCT1 to Ala through Ca2+-dependent PKCα-Akt pathway in enterocytes.
